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Interference in fluorometric hydrogen peroxide determination using scopoletin–horseradish peroxidase
Author(s) -
Donahue William F.
Publication year - 1998
Publication title -
environmental toxicology and chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.1
H-Index - 171
eISSN - 1552-8618
pISSN - 0730-7268
DOI - 10.1002/etc.5620170503
Subject(s) - scopoletin , horseradish peroxidase , chemistry , hydrogen peroxide , umbelliferone , fluorescence , fluorophore , photobleaching , peroxidase , photochemistry , fluorescence spectroscopy , biochemistry , organic chemistry , coumarin , enzyme , medicine , physics , alternative medicine , pathology , quantum mechanics
Interference in fluorometric hydrogen peroxide (H 2 O 2 ) determinations was investigated and attributed to exposure of the fluorophore scopoletin to visible light before the determination sequence. The combination of photobleaching and complex fluorometric properties of scopoletin results in unpredictable changes in its fluorescence unless the scopoletin is shielded from all excitation wavelengths for at least 3 min before determination. Addition of the enzyme horseradish peroxidase (HRP) to scopoletin solutions also causes dramatic decreases in solution fluorescence in the absence of H 2 O 2 . Use of a series of standard solutions of scopoletin, HRP, and H 2 O 2 that are shielded for 3 min before independent measurement of fluorescence is suggested for calibration to eliminate these problems in determination of nanomolar concentrations of H 2 O 2 .